Journals
Journal:
CLINICA CHIMICA ACTA
ISSN:
0009-8981
Year:
2023
Vol.:
548
pp.
117502
Background and aims: Patients' response to treatment in differentiated thyroid cancer (DTC) is classified ac-cording to serum thyroglobulin concentrations (Tg), usually using the American Thyroid Association guidelines and considering potential interfering anti-thyroglobulin antibodies (Ab-Tg). We aim to evaluate the clinical implications of changing Tg and Ab-Tg quantification method. Material and methods: Tg and Ab-Tg were quantified in 82 serum samples (60 from DTC patients) by Elecsys and Access immunoassays. Results: Elecsys immunoassay rendered higher values of Tg than Access: mean bias 5.03 ng/mL (95%CI:-14.14-24.21). In DTC patients, there was an almost perfect agreement for response classification (kappa index = 0.833). Discrepancies appeared in patients with undetermined response, with a more tendency to subclassifi-cation with Access. Ab-Tg showed a poor correlation (r = 0.5394). When Elecsys cut-off was reduced to 43 IU/ mL, agreement for positive/negative classification improved from a kappa index of 0.607 to 0.650. Prospective study with personalized follow-up showed that only 6.3% of Tg results required an analytical confirmation, being confirmed 93% of them. Conclusions: Despite the biases observed, clinical impact of an analytical change is minimal in patients' man-agement. However, cautious and personalized follow-up period after the change is still mandatory, especially in patients with Tg levels between 0.2 and 1 ng/mL.
Authors:
Lopez-Borrego, S.; Campos-Silva, C.; Sandua, Amaia; et al.
Journal:
FRONTIERS IN CELL AND DEVELOPMENTAL BIOLOGY
ISSN:
2296-634X
Year:
2023
Vol.:
10
pp.
1055288
Metastatic melanoma presents, in many cases, oncogenic mutations in BRAF, a MAPK involved in proliferation of tumour cells. BRAF inhibitors, used as therapy in patients with these mutations, often lead to tumour resistance and, thus, the use of MEK inhibitors was introduced in clinics. BRAFi/MEKi, a combination that has modestly increased overall survival in patients, has been proven to differentially affect immune ligands, such as NKG2D-ligands, in drug-sensitive vs. drug-resistant cells. However, the fact that NKG2D-ligands can be released as soluble molecules or in extracellular vesicles represents an additional level of complexity that has not been explored. Here we demonstrate that inhibition of MAPK using MEKi, and the combination of BRAFi with MEKi in vitro, modulates NKG2D-ligands in BRAF-mutant and WT melanoma cells, together with other NK activating ligands. These observations reinforce a role of the immune system in the generation of resistance to directed therapies and support the potential benefit of MAPK inhibition in combination with immunotherapies. Both soluble and EV-associated NKG2D-ligands, generally decreased in BRAF-mutant melanoma cell supernatants after MAPKi in vitro, replicating cell surface expression. Because potential NKG2D-ligand fluctuation during MAPKi treatment could have different consequences for the immune response, a pilot study to measure NKG2D-ligand variation in plasma or serum from metastatic melanoma patients, at different time points during MAPKi treatment, was performed. Not all NKG2D-ligands were equally detected. Further, EV detection did not parallel soluble protein. Altogether, our data confirm the heterogeneity between melanoma lesions, and suggest testing several NKG2D-ligands and other melanoma antigens in serum, both as soluble or vesicle-released proteins, to help classifying immune competence of patients.
Journal:
CLINICA CHIMICA ACTA
ISSN:
0009-8981
Year:
2023
Vol.:
543
pp.
117303
Aims: Characterization of PSA in extracellular microvesicles (EVs) and its reactivity to commercial methods.
Materials and methods: EVs derived from serum of 47 prostate cancer (PCa) patients, 27 benign prostatic hyperplasia (BPH) patients and 42 healthy controls were analyzed. EVs isolation and quantification of PSA immunoreactive to total (ev-T-PSA) or free (ev-F-PSA) PSA immunoassays, were performed using commercial assays. PSA in CD81+ or CD63+ EVs was determined directly in serum by an immunocapture-ELISA (IC-ELISA).
Results: Ev-T-PSA immunoreactive to Elecsys assay was detected in all samples. Median T-PSA ev/srm ratio was 2.20 % (Q1-Q3: 0.80-4.00 %), although in some samples this ratio reached 59 %. T-PSA ev/srm ratio was higher in those samples with serum T-PSA below 4 µg/L than in those exceeding that cut-off (p < 0.001). T-PSA ev/srm ratio was lower in PCa patients compared to healthy controls and BPH patients (p < 0.001). Elecsys immunoassays detected higher concentrations of ev-T-PSA and ev-F-PSA than Immulite (p < 0.001). PSA was detected by IC-ELISA more intensely in CD81+ EVs than in CD63+ EVs, and ev-T-PSA correlated with PSA+ CD63+ (p < 0.001) but not with PSA+ CD81+.
Conclusion: EVs-bound PSA is another form of circulating PSA whose measurement could be easily performed in clinical laboratories by automated immunoassays.
Journal:
CLINICAL CHEMISTRY AND LABORATORY MEDICINE
ISSN:
1434-6621
Year:
2022
Vol.:
60
N°:
7
pp.
1003 - 1010
Objectives Retrospective studies frequently assume analytes long-term stability at ultra-low temperatures. However, these storage conditions, common among biobanks and research, may increase the preanalytical variability, adding a potential uncertainty to the measurements. This study is aimed to evaluate long-term storage stability of different analytes at Methods Twenty-one analytes commonly measured in clinical laboratories were quantified in 60 serum samples. Samples were immediately aliquoted and frozen at <-70 degrees C, and reanalyzed after 11 +/- 3.9 years of storage. A change in concentration after storage was considered relevant if the percent deviation from the baseline measurement was significant and higher than the analytical performance specifications. Results Preanalytical variability (CVP) due to storage, determined by the percentage deviation, showed a noticeable dispersion. Changes were relevant for alanine aminotransferase, creatinine, glucose, magnesium, potassium, sodium, total bilirubin and urate. No significant differences were found in aspartate aminotransferase, calcium, carcinoembryonic antigen, cholesterol, C-reactive protein, direct bilirubin, free thryroxine, gamma-glutamyltransferase, lactate dehydrogenase, prostate-specific antigen, triglycerides, thyrotropin, and urea. As nonnegligible, CVP must remain included in reference change value formula, which was modified to consider whether one or two samples were frozen. Conclusions After long-term storage at ultra-low temperatures, there was a significant variation in some analytes that should be considered. We propose that reference change value formula should include the CVP when analyzing samples stored in these conditions.
Journal:
SCIENTIFIC REPORTS
ISSN:
2045-2322
Year:
2022
Vol.:
12
N°:
1
pp.
9752
Breath tests used to evaluate carbohydrates malabsorption require baseline H2 and CH4 levels as low as possible. Test cancellation is recommended when exceeding certain cut-offs (H2 ¿ 20 ppm and CH4 ¿ 10 ppm). Although following preparation protocols, many patients have baseline levels above those cut-offs. We investigated if light walking can reduce baseline H2 and CH4 levels. We retrospectively analyzed baseline H2 and CH4 levels from 1552 breath tests. Baseline levels (B1), especially in H2, were lower when obtained at later hours of the day. In those with baseline levels above cut-off, re-sampling (B2) after light walking for one hour, decreased H2 levels 8 ppm (Q1-Q3: 1-18 ppm), and 2 ppm (Q1-Q3: 0-3 ppm) for CH4. Consequently, 40% of tests with elevated B1 levels, presented B2 levels below mentioned cut-offs. Ten percent of tests considered negative when using B1 for calculations, turned positive when using B2 instead. All positive tests when using B1 values, remained elevated when using B2. Re-sampling after light walking for one hour could allow test performance in those with previous elevated baseline levels, avoiding diagnosis delays. Using the second sample for delta calculations identifies positive patients for malabsorption that would have been considered negative.
Journal:
CLINICAL CHEMISTRY
ISSN:
0009-9147
Year:
2022
Vol.:
68
N°:
9
pp.
1128 - 1132
Journal:
JOURNAL OF CANCER RESEARCH AND THERAPEUTICS
ISSN:
0973-1482
Year:
2022
Vol.:
18
N°:
4
pp.
1183 - 1185
The incidence of autoimmune thyroid disorders is higher among women with breast cancer (BC) than in other solid malignancies, while it has not a prognostic impact. Trastuzumab (T) is a humanized monoclonal antibody approved for human epidermal growth factor receptor 2 (HER2)-positive BC in the neoadjuvant, adjuvant, and metastatic scenarios. Since 2014, subcutaneous (SC) T has been employed with the same efficacy as the intravenous formulation together with an easier way of administration. To date, autoimmune thyroiditis has been linked rarely to the use of intravenous T, and no cases have been related to the SC presentation. We report two cases of HER2-positive early BC patients who developed hypothyroidism during maintenance therapy with SC T that required levothyroxine supplementation. SC T includes recombinant human hyaluronidase to facilitate tissue penetration of the drug. This enzyme may alter the thyroid gland stroma and facilitate the development of thyroid disorders. Thyroid function tests are recommended in patients on SC T.
Journal:
CYTOKINE
ISSN:
1043-4666
Year:
2021
Vol.:
141
pp.
155471
Background: Myeloid-derived suppressor cells (MDSCs) are relevant in prostate cancer microenvironment collaborating in tumor development. The main tumor marker used in this disease, prostate-specific antigen (PSA), does not provide information related to this tumor microenvironment. Cancer cells secrete exosomes carrying bioactive molecules contributing to MDSCs recruitment and induction. The aim of this study was to characterize the perioperative changes of exosomal cytokines relevant in MDSCs recruitment induced by prostatectomy in prostate cancer patients.
Methods: Blood was drawn from 26 early-stage prostate cancer patients before and after radical prostatectomy and from 16 healthy volunteers. Serum exosomes were separated by precipitation. Cytokines related with MDSC cell recruitment and activation CCL2, CXCL2, CXCL5, CXCL8, CXCL12, MIF, S100A9 and TGF-ß were measured in serum and serum-derived exosomes using immunometric assays.
Results: All cytokines were detected both in serum and exosomes, except for CXCL12, which was detected only in serum. Exosomes were enriched specially in MIF, TGF-ß and CXCL2. Presurgical MIF levels in exosomes correlated negatively with serum PSA. Also, presurgical TGF-ß decreased both in serum and exosomes as Gleason score rises. Patient¿ presurgical exosomes had increased CCL2, CXCL5 and TGF-ß levels than exosomes from healthy controls. These differences were not observed when cytokines were analyzed in serum, except for TGF-ß. Cytokine ...
Journal:
MOLECULAR THERAPY - NUCLEIC ACIDS
ISSN:
2162-2531
Year:
2021
Vol.:
25
pp.
207 - 219
Variegate porphyria (VP) results from haploinsufficiency of pro-toporphyrinogen oxidase (PPDX), the seventh enzyme in the heme synthesis pathway. There is no VP model that recapitulates the clinical manifestations of acute attacks. Combined administrations of 2-allyl-2-isopropylacetamide and rifampicin in rabbits halved hepatic PP OX activity, resulting in increased accumulation of a potentially neurotoxic heme precursor, lipid peroxidation, inflammation, and hepatocyte cytoplasmic stress. Rabbits also showed hypertension, motor impairment, reduced activity of critical mitochondrial hemoprotein functions, and altered glucose homeostasis. Hemin treatment only resulted in a slight drop in heme precursor accumulation but further increased hepatic heme catabolism, inflammation, and cytoplasmic stress. Hemin replenishment did protect against hypertension, but it failed to restore action potentials in the sciatic nerve or glucose homeostasis. Systemic porphobilinogen deaminase (PBGD) mRNA administration increased hepatic PBGD activity, the third enzyme of the pathway, and rapidly normalized serum and urine porphyrin precursor levels. All features studied were improved, including those related to critical hemoprotein functions. In conclusion, the VP model recapitulates the biochemical characteristics and some clinical manifestations associated with severe acute attacks in humans.
Authors:
Sendino, T.; Sandúa, A.; Calleja, S.; et al.
Journal:
ADVANCES IN LABORATORY MEDICINE / AVANCES EN MEDICINA DE LABORATORIO
ISSN:
2628-491X
Year:
2020
Vol.:
1
N°:
4
pp.
1 - 5
Journal:
CLINICAL CHEMISTRY AND LABORATORY MEDICINE
ISSN:
1434-6621
Year:
2020
Vol.:
58
N°:
8
pp.
1341 - 1348
Background: Genomic alterations studies in cell-free DNA (cfDNA) have increasing clinical use in oncology. Nextgeneration sequencing (NGS) technology provides the most complete mutational analysis, but nowadays limited data are available related to the comparison of results reported by different platforms. Here we compare two NGS panels for cfDNA: OncomineT Pan-Cancer Cell-Free Assay (Thermo Fisher Scientific), suitable for clinical laboratories, and Guardant360 (R) (GuardantHealth), with more genes targeted but only available in an outsourcing laboratory.
Methods: Peripheral blood was obtained from 16 advanced cancer patients in which Guardant360 (R) (G360) was requested as part of their clinical assistance. Blood samples were sent to be analyzed with G360 panel, and an additional blood sample was drawn to obtain and analyze cfDNA with OncomineT Pan-Cancer (OM) panel in an Ion GeneStudio S5T System.
Results: cfDNA analysis globally rendered 101 mutations. Regarding the 55/101 mutations claimed to be included by manufacturers in both panels, 17 mutations were reported only by G360, 10 only by OM and 28 by both. In those coincident cases, there was a high correlation between the variant allele fractions (VAFs) calculated with each panel (r = 0.979, p < 0.01). Regarding the six actionable mutations with an FDA-approved therapy reported by G360, one was missed with OM. Also, 12 mutations with clinical trials available were reported by G360 but not by OM.
Conclusions: In summary, G360 and OM can produce different mutational profile in the same sample, even in genes included in both panels, which is especially important if these mutations are potentially druggable.
Journal:
ADVANCES IN LABORATORY MEDICINE / AVANCES EN MEDICINA DE LABORATORIO
ISSN:
2628-491X
Year:
2020
Vol.:
1
N°:
1
pp.
20190017
Background: Thyroglobulin (Tg) is fundamental for
differentiated thyroid cancer (DTC) monitoring. Tg detection can be enhanced using recombinant human thyroidstimulating hormone (TSH) (rhTSH). This study is aimed to
evaluate the use of the rhTSH stimulation test when using a
high-sensitivity Tg assay.
Methods: We retrospectively studied 181 rhTSH tests from
114 patients with DTC and negative for antithyroglobulin
antibodies (anti-TgAb). Image studies were performed in
all cases. Serum Tg and anti-TgAb were measured using
specific immunoassays.
Results: rhTSH stimulation in patients with basal serum Tg
(b-Tg) concentrations lower than 0.2 ng/mL always resulted in
rhTSH-stimulated serum Tg (s-Tg) concentrations lower than
1.0 ng/mL and negative structural disease. In patients with bTg concentration between 0.2 and 1.0 ng/mL, s-Tg detected
one patient (1/30) who showed biochemical incomplete
response. Patients with negative images had lower s-Tg than
Journal:
CLINICAL CHEMISTRY AND LABORATORY MEDICINE
ISSN:
1434-6621
Year:
2019
Vol.:
57
N°:
10
pp.
1539 - 1545
Background: Exosomes are nanovesicles released by cells that can be detected in blood. Exosomes contain several molecules, such as cytokines that have potential utility as disease biomarkers. The aim of the present work is to compare six different commercial kits suitable for the clinical laboratory in relation to the efficiency and purity of exosome isolation, and their effect in subsequent cytokines analysis. Methods: Serum exosomes were obtained from 10 volunteers using six commercial kits: exoEasy, ExoQuick, Exo-spin, ME kit, ExoQuick Plus and Exo-Flow. Exosome concentrations and size distributions were quantified by nanoparticle tracking analysis. Exosome markers CD63, CD9 and TSG101 were determined by Western blot. ApoB and albumin were measured using nephelometry. S100A9, CXCL5 and CXCL12 were measured using a Luminex assay. Results: The concentration of particles obtained between different kits varied by a factor of 100. There was no correlation in particle concentrations extracted between different kits, except between ExoQuick and Exo-Flow. The highest exosome purity was achieved with ExoQuick Plus and exoEasy, while the lowest were achieved with ME and ExoQuick. Albumin was present in all exosome extracts analyzed and ApoB in all except those extracted with Exo-Flow and ME. Cytokine detection varied depending on the purification kit used and there was no correlation in cytokine concentrations between samples obtained with different kits. Conclusions: Both the sample and the type of commercial kit used affect the efficiency and purity of exosome isolation. In addition, the exosome purification method deeply affects the capability to detect and quantify cytokines.
Journal:
DISEASE MARKERS
ISSN:
0278-0240
Year:
2019
Vol.:
2019
N°:
7954921
pp.
1 - 7
Epidermal growth factor receptor (EGFR) mutational testing in advanced non-small-cell lung cancer (NSCLC) is usually performed in tumor tissue, although cfDNA (cell-free DNA) could be an alternative. We evaluated EGFR mutations in cfDNA as a complementary tool in patients, who had already known EGFR mutations in tumor tissue and were treated with either EGFR-tyrosine kinase inhibitors (TKIs) or chemotherapy. We obtained plasma samples from 21 advanced NSCLC patients with known EGFR tumor mutations, before and during therapy with EGFR-TKIs and/or chemotherapy. cfDNA was isolated and EGFR mutations were analyzed with the multiple targeted cobas EGFR Mutation Test v2. EGFR mutations were detected at baseline in cfDNA from 57% of patients. The semiquantitative index (SQI) significantly decreased from the baseline (median = 11, IQR = 9.5-13) to the best response (median = 0, IQR = 0-0, p < 0.01), followed by a significant increase at progression (median = 11, IQR = 11-15, p < 0.01) in patients treated with either EGFR-TKIs or chemotherapy. The SQI obtained with the cobas EGFR Mutation Test v2 did not correlate with the concentration in copies/mL determined by droplet digital PCR. Resistance mutation p.T790M was observed at progression in patients with either type of treatment. In conclusion, cfDNA multiple targeted EGFR mutation analysis is useful for treatment monitoring in tissue of EGFR-positive NSCLC patients independently of the drug received.
Journal:
ADVANCES IN LABORATORY MEDICINE / AVANCES EN MEDICINA DE LABORATORIO
ISSN:
2628-491X
Year:
2019
Vol.:
1
N°:
2
pp.
1 - 2
Journal:
ANNALS OF TRANSLATIONAL MEDICINE
ISSN:
2305-5839
Year:
2016
Vol.:
4
N°:
24
pp.
504
Journal:
CLINICA CHIMICA ACTA
ISSN:
0009-8981
Year:
2016
Vol.:
454
pp.
28 - 32
Background: Malignant melanoma is an aggressive cancer with an increasing incidence. Exosomes are actively secreted microvesicles, whose characteristics reflect those of the cell they are originated in. The aim of this study was to identify and evaluate the presence of the melanoma biomarlcers MIA, S100B and tyrosinase-related protein 2 (TYRP2) in exosomes and their potential clinical utility.
Methods: Serum samples were obtained from stage IV melanoma patients, melanoma-free patients and healthy controls. Exosomes were precipitated and TYRP2, MIA and S100B concentrations were quantified in serum, exosomes, and exosome-free serum.
Results: Both MIA and S100B were detected in exosomes and correlated significantly with serum concentrations (S100B: r = 0.968; MIA: r = 0.799; p < 0.001). MIA and S100B concentrations in exosomes were significantly higher in melanoma patients than in healthy controls and disease-free patients. However, TYRP2 concentrations in exosomes did not differ between these three groups. ROC curves analysis rendered AUCs for MIA of 0.883 (p < 0.01) and of 0.840 for S100B (p < 0.01). Patients with exosome MIA concentration higher than 2.5 mu g/L showed shorter median survival related to those with lower level (4 versus 11 months; p < 0.05).
Conclusions: MIA and S100B can be detected in exosomes from melanoma patients and their quantification presents diagnostic and prognostic utility.
Journal:
TUMOR BIOLOGY
ISSN:
1010-4283
Year:
2016
Vol.:
37
N°:
10
pp.
13687 - 13694
Mutation analysis of epidermal growth factor receptor (EGFR) gene is essential for treatment selection in non-small cell lung cancer (NSCLC). Analysis is usually performed in tumor samples. We evaluated the clinical utility of EGFR analysis in plasma cell-free DNA (cfDNA) from patients under treatment with EGFR inhibitors. We selected 36 patients with NSCLC and EGFR-activating mutations. Blood samples were collected at baseline and during treatment with EGFR inhibitors. Wild-type EGFR, L858R, delE746-A750, and T790M mutations were quantified in cfDNA by droplet digital PCR. Stage IV patients had higher total circulating EGFR copy levels than stage I (3523 vs. 1003 copies/mL; p < 0.01). There was high agreement for activating mutations between baseline cfDNA and tumor samples, especially for L858R mutation (kappa index = 0.679; p = 0.001). In 34 % of advanced NSCLC patients, we detected mutations in cfDNA not previously detected in tumor samples and double mutations in 17 %. Patients with baseline total EGFR copy levels above the median presented decreased overall survival (OS) (341 vs. 870 days, p < 0.05) and progression-free survival (PFS) (238 vs. 783 days; p < 0.05) compared with those with total EGFR copy levels below the median. Patients with baseline concentrations of activating mutations above the median (94 copies/mL) had lower OS (317 vs. 805 days; p < 0.05) and PFS (195 vs. 724 days; p < 0.05). During follow-up, T790M resistance mutation was detected in 53 % of patients. Total and mutated EGFR analysis in cfDNA seems a relevant tool to characterize the molecular profile and prognosis of NSCLC patients harboring EGFR mutations.
Journal:
CLINICA CHIMICA ACTA
ISSN:
0009-8981
Year:
2014
Vol.:
429
pp.
168 - 174
BRAF V600 mutation has been reported in more than 50% of melanoma cases and its presence predicts clinical activity of BRAF inhibitors (iBRAF). We evaluated the rote of MIA, S100 and LDH to monitor iBRAF efficiency in advanced melanoma patients presenting BRAF V600 mutations. This was a prospective study of melanoma patients harboring the BRAF V600 mutation and treated with iBRAF within a clinical trial (dabrafenib) or as part of an expanded access program (vemurafenib). MIA, S100 and LDH were analyzed in serum at baseline, and every 4-6 weeks during treatment. Eighteen patients with melanoma stages IIIc-IV were enrolled with 88.8% of response rate to iBRAF. Baseline concentrations of all the tumor markers correlated with tumor burden. MIA and S100 concentrations decreased significantly one month after the beginning of treatment and, upon progression, their concentrations increased significantly above the minimum levels previously achieved. MIA levels lower than 9 mu g/L one month after the beginning of treatment and S100 concentrations lower than 0.1 mu g/L at the moment of best response were associated With improved progression-free survival. In conclusion, MIA and S100 are useful to monitor response in melanoma patients treated with iBRAF.
Journal:
ARCHIVES OF PATHOLOGY AND LABORATORY MEDICINE
ISSN:
0003-9985
Year:
2014
Vol.:
138
N°:
6
pp.
828 - 832
CONTEXT:
Malignant melanoma is an aggressive tumor that produces exosomes, which contain microRNAs (miRNAs) that could be of utility in following tumoral cell dysregulation. MicroR-125b is a miRNA whose down-regulation seems to be implicated in melanoma progression.
OBJECTIVE:
To analyze miR-125b levels in serum, and in exosomes obtained from serum, from patients with advanced melanoma.
DESIGN:
Serum samples were obtained from 21 patients with advanced melanoma, from 16 disease-free patients with melanoma, and from 19 healthy volunteers. Exosomes were isolated from serum by precipitation, and miR-16 and miR-125b levels were quantified by real-time polymerase chain reaction.
RESULTS:
MicroR-16, but not miR-125b, was detected in all samples, and miR-16 levels were significantly higher in serum than they were in exosomes. MicroR-16 expression levels did not differ significantly between the 2 groups (patients with melanoma and healthy donors). There was a significant relationship between miR-125b and miR-16 levels in exosomes. Additionally, miR-125b levels in exosomes were significantly lower in patients with melanoma compared with disease-free patients with melanoma and healthy controls.
CONCLUSIONS:
Exosomes can provide a suitable material to measure circulating miRNA in melanoma, and miR-16 can be used as an endogenous normalizer. Lower levels of miR-125b in exosomes obtained from serum are associated with advanced melanoma disease, probably reflecting the tumoral cell dysregulation.
Journal:
EUROPEAN JOURNAL OF IMMUNOLOGY
ISSN:
0014-2980
Year:
2013
Vol.:
43
N°:
7
pp.
1933-9
The nonclassical human leukocyte antigen-G (HLA-G) is a tolerogenic molecule that can be released to the circulation by expressing cells. This molecule can form dimers but some other complexed HLA-G forms have been proposed to be present in vivo. Here, we further characterized these other complexed HLA-G forms in vivo. Ascitic and pleural exudates from patients were selected based on positivity for HLA-G by ELISA. Complexed HLA-G was detected in exosomes, which indicates an intracellular origin of these forms. 2D-PAGE analysis of exudates and isolated exosomes showed that these high molecular weight complexes were more heterogeneous than the HLA-G1 expressed by cell cultures. Treatment with deglycosylating enzymes did not change the molecular weight of HLA-G complexes. Immunoblot analysis of exudates and exosomes with an anti-ubiquitin antibody showed that at least some of these structures correspond to ubiquitinated HLA-G. HLA-G ubiquitination could be reproduced in vitro in HLA-G1-transfected cell lines, although with a lower modified/nonmodified protein proportion than in exudates. In summary, we demonstrate new circulating HLA-G forms in vivo that open a new perspective in the study of HLA-G function and analysis
Authors:
HowangYin, K.Y.; Loustau, M.; Wu, J.; et al.
Journal:
CELLULAR AND MOLECULAR LIFE SCIENCES
ISSN:
1420-682X
Year:
2012
Vol.:
69
N°:
23
pp.
4041 - 4049
The non-classical Human leukocyte antigen G (HLA-G) differs from classical HLA class I molecules by its low genetic diversity, a tissue-restricted expression, the existence of seven isoforms, and immuno-inhibitory functions. Most of the known functions of HLA-G concern the membrane-bound HLA-G1 and soluble HLA-G5 isoforms, which present the typical structure of classical HLA class I molecule: a heavy chain of three globular domains alpha(1)-alpha(2)-alpha(3) non-covalently bound to beta-2-microglobulin (B2M) and a peptide. Very little is known of the structural features and functions of other HLA-G isoforms or structural conformations other than B2M-associated HLA-G1 and HLA-G5. In the present work, we studied the capability of all isoforms to form homomultimers, and investigated whether they could bind to, and function through, the known HLA-G receptors LILRB1 and LILRB2. We report that all HLA-G isoforms may form homodimers, demonstrating for the first time the existence of HLA-G4 dimers. We also report that the HLA-G alpha(1)-alpha(3) structure, which constitutes the extracellular part of HLA-G2 and HLA-G6, binds the LILRB2 receptor but not LILRB1. This is the first report of a receptor for a truncated HLA-G isoform. Following up on this finding, we show that the alpha(1)-alpha(3)-Fc structure coated on agarose beads is tolerogenic and capable of prolonging the survival of skin allografts in B6-mice and in a LILRB2-transgenic mouse model. This study is the first proof of concept that truncated HLA-G isoforms could be used as therapeutic agents.
Journal:
CRITICAL REVIEWS IN CLINICAL LABORATORY SCIENCES
ISSN:
1040-8363
Year:
2012
Vol.:
49
N°:
3
pp.
63 - 84
Journal:
TUMOR BIOLOGY
ISSN:
1010-4283
Year:
2011
Vol.:
32
N°:
6
pp.
1155 - 1161
Journal:
Clinical Chemistry (Baltimore)
ISSN:
0009-9147
Year:
2011
Vol.:
57
N°:
7
pp.
1013 - 1022
Journal:
CELLULAR AND MOLECULAR LIFE SCIENCES
ISSN:
1420-682X
Year:
2010
Vol.:
67
N°:
7
pp.
1133 - 1145
Trogocytosis is the uptake of membranes from one cell by another. Trogocytosis has been demonstrated for monocytes, B cells, T cells, and NK cells. The acquisition of the tolerogenic molecule HLA-G by T cells and NK cells makes them behave as regulatory cells. We investigated here whether HLA-G, which is expressed by tumor cells in vivo, could be acquired by monocytes and if this transfer could have functional consequences. We demonstrate that resting, and even more so, activated monocytes efficiently acquire membrane-bound HLA-G from HLA-G tumor cells by trogocytosis. However, we demonstrate that HLA-G quickly disappears from the surface of the monocytes in contrast to the HLA-G acquired by T cells. Consequently, HLA-G(acq+) monocytes do not reliably inhibit the on-going proliferation of autologous activated T cells and do not inhibit their cytokine production. Thus, we show that the acquirer cell may control the functional outcome of trogocytosis.
Journal:
American Journal of Reproductive Immunology
ISSN:
1046-7408
Year:
2010
Vol.:
64
N°:
5
pp.
367 - 74
Journal:
Cell Research
ISSN:
1001-0602
Year:
2010
Vol.:
20
N°:
11
pp.
1239 - 1251
